Our goal is to develop a screening methodology that can be used to rapidly and efficiently identify small molecules that disrupt protein interactions and inhibit the function of the protein. The methodology will also provide critical information regarding the binding site of the molecule on the target protein. As proof-of- principle, we identified a disrupter of a dimeric viral protease in the herpes virus family that has important clinical relevance yet has thus far proven to be an intractable target. Numerous viral protease drug targets are dimeric, including those in the herpes virus family and could also be inhibited by dimer disruption. We have previously shown that induced structure of a helical switch at the dimer interface regulates activity of the Kaposi's sarcoma herpes virus (KSHV) protease using a combination of chemical biology and NMR. To test our proposed screening paradigm we will carry out the following specific aims: Aim I. Adapt and validate existing fluorescence-based protease assays for high throughput screening (HTS) format to identify active site inhibitors and dimer disrupters. Aim II. Screen more than 200,000 compounds from several high potential libraries for KSHV protease inhibitors using HTS fluorescence assays and identify allosteric inhibitors of herpes virus proteases. Aim III. Characterize binding sites of hits identified in Aim II using 2D NMR of 13C-Met labeled protease and determine the binding sites of novel inhibitors using 2D NMR of 15N-specifically labeled protease. At the completion, we will have demonstrated a robust proof-of-principle in an oligomeric enzyme system and will have identified a small subset of inhibitory molecules. Some of these "hits" will be specific for a family member while others will be broad-spectrum inhibitors of the entire family. All will be potent (<microM), and will be characterized in terms of their binding sites (active site, dimer interface, or allosteric site) for subsequent development. Collaborations are in place to provide libraries that are biased for success. Our long-term goal is to develop a general strategy for modulating protein interactions and function with small drug-like molecules by targeting allosteric sites. The techniques developed here will have general applicability to many soluble protein drug targets, which previously have proven intractable to drug discovery efforts and will offer a new approach to drug discovery, opening up new therapeutic avenues.